Methods for analysis of Ca2+/H+ antiport activity in synaptic vesicles isolated from sheep brain cortex

Abstract

The involvement of Ca2+-storage organelles in the modulation of synaptic transmission is well-established [M.K. Bennett, Ca2+ and the regulation of neurotransmitter secretion, Curr. Opin. Neurobiol. 7 (1997) 316-322 [1]; M.J. Berridge, Neuronal calcium signaling, Neuron 21 (1998) 13-26 [2]; Ph. Fossier, L. Tauc, G. Baux, Calcium transients and neurotransmitter release at an identified synapse, Trends Neurosci. 22 (1999) 161-166 [7]]. Various Ca2+ sequestering reservoirs (mitochondria, endoplasmic reticulum and synaptic vesicles) have been reported at the level of brain nerve terminals [P. Kostyuk, A. Verkhratsky, Calcium stores in neurons and glia, Neuroscience 63 (1994) 381-404 [18]; V. Mizuhira, H. Hasegawa, Microwave fixation and localization of calcium in synaptic terminals using X-ray microanalysis and electron energy loss spectroscopy imaging, Brain Res. Bull. 43 (1997) 53-58 [21]; A. Parducz, Y. Dunant, Transient increase of calcium in synaptic vesicles after stimulation, Neuroscience 52 (1993) 27-33 [23]; O.H. Petersen, Can Ca2+ be released from secretory granules or synaptic vesicles?, Trends Neurosci. 19 (1996) 411-413 [24]]. However, the knowledge of the specific contribution of each compartment for spatial and temporal control of the cytoplasmic Ca2+ concentration requires detailed characterization of the Ca2+ uptake and Ca2+ release mechanisms by the distinct intracellular stores. In this work, we described rapid and simple experimental procedures for analysis of a Ca2+/H+ antiport system that transport Ca2+ into synaptic vesicles at expenses of the energy of a [Delta]pH generated either by activity of the proton pump or by a pH jumping of the vesicles passively loaded with protons. This secondary active Ca2+ transport system requires high Ca2+ concentrations (>100 [mu]M) for activation, it is dependent on the chemical component ([Delta]pH) of the proton electrochemical gradient across the synaptic vesicle membrane and its selectivity is essentially determined by the size of the transported cation [P.P. Gonçalves, S.M. Meireles, C. Gravato, M.G.P. Vale, Ca2+-H+-Antiport activity in synaptic vesicles isolated from sheep brain cortex, Neurosci. Lett. 247 (1998) 87-90 [10]; P.P. Gonçalves, S.M. Meireles, P. Neves, M.G.P. Vale, Ionic selectivity of the Ca2+/H+ antiport in synaptic vesicles of sheep brain cortex, Mol. Brain Res. 67 (1999) 283-291 [11]; P.P. Gonçalves, S.M. Meireles, P. Neves, M.G.P. Vale, Synaptic vesicle Ca2+/H+ antiport: dependence on the proton electrochemical gradient, Mol. Brain Res. 71 (1999) 178-184 [12]]. The protocols described here allow to ascertain the characteristics of the Ca2+/H+ antiport in synaptic vesicles and, therefore, may be useful for clarification of the physiological role of synaptic vesicles in fast buffering of Ca2+ at various sites of the neurotransmission machinery.Theme: Excitable membranes and synaptic transmission.Topic: Presynaptic mechanisms.